The production of recombinant proteins for therapeutic, nutraceutical or industrial uses has enjoyed great success over the past several decades. Introduction of heterologous genes having desired nucleotide sequences into a variety of expression hosts is now routine. This process nearly always leads to expression of polypeptides or proteins having the correct predicted primary amino acid residue sequence (primary structure) encoded by the introduced nucleotides. In many instances, however, the protein or polypeptide that is ultimately produced can possess the correct primary amino acid residue sequence of the naturally-produced molecule, but lack the biological activity expected of that material.
Biological activity, given the proper primary structure of the expressed product, can be a function of the protein's secondary, tertiary or quaternary structure. These structural features include having the proper folding and internal hydrogen, Van der Waals, ionic and disulfide bonding patterns, appropriate intermolecular and intramolecular subunit interactions, and also having the proper post-translational modifications, as for instance glycosylation. For example, disulfide bond formation occurs spontaneously in the lumen of the endoplasmic reticulum (ER) of eukaryotic cells, but not in the reducing environment of the cytosol of prokaryotes, which makes bacterial cells such as Escherichia coli poor hosts for the synthesis of correctly-folded mammalian proteins that are normally stabilized by disulfide bonds. Disulfide bond formation can occur in the periplasmic space of E. coli where certain prokaryotic chaperoning, foldases and PDI-like proteins are functional (Fernandez, et al., 2001. Mol. Microbiol. April 40(2):332-346). However, even in this compartment the bacterial oxi-redox system is not very efficient for eukaryotic proteins.
A particular case in point relates to erythropoietin (EPO), a protein that stimulates red blood cell production. Recombinant EPO is disclosed in Lin, U.S. Pat. No. 4,703,008, which describes activities for heterologous human EPO protein expressed from E. coli, S. cerevisiae, and mammalian Chinese hamster ovary (CHO) and African green monkey kidney (COS-1) cells. Although EPO expressed by each cell type had the correct primary amino acid sequence and was cross-reactive with anti-EPO antisera, only the proteins expressed from mammalian cells exhibited the expected levels of biological activity as determined by in vitro and in vivo assays. These observed differences in biological activity were determined to be a function of improper glycosylation in the prokaryotic and lower eukaryotic host cells. E. coli, a prokaryote, does not perform the eukaryotic enzymatic steps of N-linked glycosylation. Yeast cells are eukaryotes and capable of N-linked glycosylation, but their glycosylation enzymes differ from that of animals and plants and consequently result in a different pattern of terminal glycosylation for secreted proteins. On the other hand, the CHO and COS-1 cells used to provide proteins of substantially correct biological activity were mammalian, and the proteins expressed therefrom were consequently useful. Published studies of glycosylated and aglycosylated EPO indicate that glycosylation plays a critical role in stabilizing erythropoietin under denaturing conditions (Narhi et al., (1991) J. Biol. Chem. 266(34):23022-23026). In addition, it has been reported that in vivo life time and activity of EPO can be related to the glycosylation state of the molecule, and correct interaction with the erythrogenic EPO receptor is also affected by EPO glycosylation pattern.
Eukaryotic cells are therefore greatly preferred for recombinant production of therapeutic, industrial and other useful proteins of eukaryotic origin. Consequently, many different types of eukaryotic cells and organisms have been shown to be capable of producing biologically active recombinant proteins. Unfortunately, many such eukaryotic expression systems are inefficient with respect to protein product yield and cost of manufacture, even when proteins are secreted extracellularly. The high costs frequently derived from low recombinant protein production levels and/or from complicated downstream protein isolation and purification procedures can invalidate a protein's commercial application. Active research is thus being done to improve both production levels and purification procedures.
One way of improving the efficiency of recombinant protein isolation is by means of intracellular concentration. One of these approaches is the random aggregation of recombinant proteins into non-secreted inclusion bodies which can be separated from lysed cells by density-based purification techniques. Insoluble inclusion bodies are amorphous protein deposits found in bacteria expressing complex recombinant proteins (such as those of eukaryotic origin). The absence of specialized eukaryotic molecular chaperones in prokaryotic cells results in random folding of eukaryotic proteins. Structural characterization studies have shown that the insoluble nature of inclusion bodies may be due to the random hydrophobic intermolecular interactions of proteins which are not correctly folded (Seshadri et al., 1999, Methods Enzymol. 309:559-576). The general strategy used to recover active proteins from inclusion bodies subsequent to their separation from cell material requires the complete solubilization of the recombinant protein to disrupt the random aggregates followed by one or more chemical refolding steps. This is an important issue because the efficiency of protein renaturation is highly limiting, particularly if the protein contains disulfide bonds (Clarc, Ed., April 2001 Curr. Opin. Biotechnol. 12(2):202-207).
More particularly, high concentrations of strong denaturants and chaotropic agents (e.g. detergents, urea and guanidinium hydrochloride) are required for solubilization of the aggregated and unfolded proteins in inclusion bodies. These agents must be dialyzed away completely in order to later refold the proteins into their correct and biologically active conformations. As a consequence the yield of correctly refolded recombinant proteins from inclusion bodies is extremely low, and moreover the biological activities of such refolded proteins are typically much less than that of the native-formed proteins.
Protein bodies (PBs) are naturally-occurring structures in certain plant seeds that have evolved to concentrate storage proteins intracellularly in eukaryotic cells while retaining correct folding and biological activity. PBs share some of the characteristics of the inclusion bodies from bacteria. They are dense, and contain a high quantity of proteins that are tightly packed by hydrophobic interactions [Momany et al., 2006 J. Agric. Food Chem. January 25; 54(2):543-547 and Garrat, et al., 1993 Proteins January; 15(1):88-99]. However, in contrast to the randomly-aggregated proteins in bacterial inclusion bodies, the proteins in PBs are thought to be aggregated in a non-random (assembled) manner.
A new technology for creation of synthetic PBs based on the fusion of a plant seed storage protein domain with a heterologous protein of interest (WO 2004/003207) has been developed to increase the stability and accumulation of recombinant proteins in higher plants. These storage proteins are specific to plant seeds wherein they accumulate stably in natural PBs (Galili et al., 1993, Trends Cell Biol 3:437-442) following insertion into the lumen of the ER via a signal peptide and assembly into ER-derived protein bodies (ER-PBs) (Okita et al., 1996 Annu. Rev. Plant Physiol Mol. Biol. 47:327-350; Herman et al., 1999 Plant Cell 11:601-613; Sanderfoot et al., 1999 Plant Cell 11:629-642). Full-length recombinant storage proteins have also been observed to assemble into PB-like organelles in non-plant host systems as Xenopus oocytes following injection of the corresponding mRNAs. This system has been used as a model to study the targeting properties of these storage proteins (Simon et al., 1990, Plant Cell 2:941-950; Altschuler et al., 1993, Plant Cell 5:443-450; Torrent et al., 1994, Planta 192:512-518) and to test the possibility of modifying the 19 kDa α-zein, a maize prolamin, by introducing the essential amino acids lysine and tryptophan into its sequence, without altering its stability (Wallace et al, 1988, Science 240:662-664).
Zeins, the complex group of maize prolamins, have also been produced recombinantly in yeast. Coraggio et al. (1988, Eur J Cell Biol 47:165-172), expressed native and modified α-zeins in yeast to study targeting determinants of this protein. Kim et al., 2002, Plant Cell 14: 655-672, studied the possible α-, β-, γ- and δ-zein interactions that could lead to protein body formation. To address this question, they transformed yeast cells with cDNAs encoding these proteins. In addition, those authors constructed zein-GFP fusion proteins to determine the subcellular localization of zein proteins in yeast cells but did not observe formation of dense, concentrated structures characteristic of bona fide PBs. It is worth to noting that Kim et al. (2002, Plant Cell 14: 655-672) concluded that yeast is a poor model for the study of zein interactions because zeins accumulated very poorly in transformed yeast. Yeast has also been used as a model to study the mechanisms that control the transport and deposition of gliadin storage proteins in wheat (Rosenberg et al., 1993, Plant Physiol 102:61-69).
These results in yeast as well as the similarities between bacterial inclusion bodies and PBs suggested that proteins accumulated in synthetic PBs would not be active unless renaturation steps were performed. Moreover, the presence of disulfide bonds in some natural PB-assembling protein domains, as for instance RX3, [Ludevid et al., 1984 Plant Mol. Biol. 3:227-234 and Kawagoe et al., 2005 Plant Cell April 17(4):1141-1153], which are probably involved in PB formation and stabilization, could represent an additional difficulty for production of a biologically active, native-folded protein in PBs. This would be particularly relevant for a recombinant protein that contains its own cysteine residues that might interact inappropriately with cysteines in the PB-assembling domain. The observation of biological activity without the need for refolding and renaturation of a wide variety of proteins produced in synthetic PBs in non-yeast eukaryotic hosts was therefore unexpected.
Biological activity is particularly relevant for vaccines, which must induce a correct immune response in an immunized human or other animal. Several new vaccines are composed of synthetic, recombinant, or highly purified subunit immunogens (antigens) that are thought to be safer than whole-inactivated or live-attenuated vaccines. However, the absence of immunomodulatory components having adjuvant properties associated with attenuated or killed vaccines often results in weaker immunogenicity for such vaccines.
Immunologic adjuvants are agents that enhance specific immune responses. An immunologic adjuvant can be defined as any substance or formulation that, when incorporated into a vaccine, acts generally to accelerate, prolong, or enhance the quality of specific immune responses to vaccine antigens. The word adjuvant is derived from the Latin verb adjuvare, which means to help or aid. Adjuvant mechanisms of action include the following: (1) increasing the biological or immunologic half-life of vaccine immunogens; (2) improving antigen delivery to antigen-presenting cells (APCs), as well as antigen processing and presentation by the APCs; and (3) inducing the production of immunomodulatory cytokines.
Phagocytosis involves the entry of large particles, such as apoptotic cells or whole microbes. The capacity of the cells to engulf large particles likely appeared as a nutritional function in unicellular organisms; however complex organisms have taken advantage of the phagocytic machinery to fulfill additional functions. For instance, the phagocytosis of antigens undertaken by the macrophages, the B-cells or the dendritic cells represents a key process in innate and adaptive immunity. Indeed, phagocytosis and the subsequent killing of microbes in phagosomes form the basis of an organism's innate defense against intracellular pathogens. Furthermore, the degradation of pathogens in the phagosome lumen and the production of antigenic peptides, which are presented by phagocytic cells to activate specific lymphocytes, also link phagocytosis to adaptive immunity (Jutras et al., 2005, Annual Review in Cell Development Biology. 21:511-27).
The proteins present on engulfed particles encounter an array of degrading proteases in phagosomes. Yet, this destructive environment generates peptides that are capable of binding to MHC class II molecules. Newly formed antigen-MHC class II complexes are delivered to the cell surface for presentation to CD4+ T cells (Boes et al. 2002 Nature 418:983-988). The activation of these cells induces the Th2 subset of cytokines such as IL-4 and IL-5 that help B cells to proliferate and differentiate, and is associated with humoral-type immune response.
A large body of evidence indicates that, in addition to the clear involvement of the MHC class II pathway in the immune response against phagocytosed pathogens, antigens from pathogens, including mycobacteria, Salmonella, Brucella, and Leishmania, can elicit an antigen cross-presentation. That is to say, the presentation of engulfed antigen by phagocytosis by the MHC class I-dependent response promotes the proliferation of CD8+ cytotoxic T cells (Ackerman et al., 2004 Nature Immunology 5(7):678-684; Kaufmann et al., 2005 Current Opinions in Immunology 17(1):79-87).
Dendritic cells play a central antigen presentation role to induce the immune system (Blander et al., Nature Immunology 2006 10:1029-1035). Although rare, dendritic cells are the most highly specialized APC, with the ability both to instigate and regulate immune reactivity (Lau et al. 2003 Gut 52:307-314). Although dendritic cells are important in presenting antigens, particularly to initiate primary immune responses, macrophages are the APC type most prominent in inflammatory sites and are specialized for clearing necrotic and apoptotic material. Macrophages can act not only as APCs, but can also perform either pro- or anti-inflammatory roles, dependent on the means by which they are activated.
Considering that APCs play a central role in the induction and regulation of the adaptive immunity (humoral and cellular), the recognition and phagocytosis of an antigen by those cells can be considered a key step in the immunization process. A wide variety of techniques based on the uptake of fluorescent particles have been developed to study phagocytosis by the macrophages (Vergne et al., 1998 Analytical Biochemistry 255:127-132).
An important aspect in veterinary vaccines is the genetic diversity of the species being considered and the requirement for generic systems that work across different species. To a large degree, this diversity limits the use of molecular targeting techniques to cell surface markers and immune modulators such as cytokines, because for many species including wildlife, only minimal knowledge of these molecules is available. Thus, adjuvants that rely on universal activation signals of the innate immune response (i.e. that are identical in different species) are to be preferred. Taking these requirements into consideration, particulate vaccine delivery systems are well suited for veterinary and wildlife vaccine strategies (Scheerlinck et al., 2004 Methods 40:118-124).
As is discussed in greater detail hereinafter, the present invention discloses that the expression of a fusion protein comprised of (i) a protein sequence that mediates induction of recombinant protein body-like assemblies (RPBLAs) linked to (ii) a biologically active polypeptide (protein of interest or target) induces the accumulation of those RPBLAs in cells of eukaryotic organisms such as plants, fungi, algae and animals, producing a biologically active target (protein).